Defects in the core human mismatch repair (MMR) genes are the cause of Lynch syndrome (LS/HNPCC), as well as 10-40% of sporadic colorectal, gastric, endometrial, ovarian and upper urinary tract tumors. MMR recognizes and repairs polymerase misincorporation errors, suppresses recombination between non-allelic partially homologous DNA sequences during double-stranded break (DSB) repair (homeologous recombination; HEOR), and functions as a lesion sensor in DNA damage signaling. Unrepaired errors in MMR-deficient cells lead to mutations (Mutator Phenotype) that drive tumorigenesis. Importantly, MMR- deficient tumors display resistance to several common chemotherapeutic drugs. The components of MMR from numerous organisms have been purified and MMR reactions have been reconstituted in vitro. Approximately a dozen fundamental biochemical properties have been detailed that together may account for the excision of a mismatch from DNA. Based on these studies several mechanisms for the MMR reaction have been proposed. Remarkably, the biochemical, genetic and structural studies performed to date have not fuly determined the function(s) of the core MMR components or resolved the validity of the proposed mechanisms for MMR. Moreover, the vast majority of biochemical studies have been performed with naked DNA that is significantly different from the cellular chromatin where MMR functions occur. We have developed three robust real-time single molecule measures capable of visualizing and interrogating the detailed function(s) of MMR components. Using these new methodologies we propose to resolve the mechanism of MMR on naked DNA and then determine the function(s) of MMR components on physiologically relevant chromatin for comparison. In this renewal application we propose the following Specific Aims: 1.) enumerate the precise mechanism of human MMR using single molecule analysis, 2.) determine the functions of the human MMR proteins on physiologically relevant chromatin, and 3.) examine the biophysical mechanism of homeologous recombination suppression by MMR. We will apply our results to determine the functional defects associated with missense mutation found in LS/HNPCC families. These studies should provide a visual and quantitative platform for understanding the detailed function(s) of core MMR components in repair, damage signaling, drug resistance and tumorigenesis PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE More than 700 unique genetic alterations of the human mismatch repair (MMR) genes have been reported in families susceptible to LS/HNPCC (://www.hgmd.cf.ac.uk), of which 20-75% are missense alterations with unknown functional consequences. This proposal will further develop and utilize inovative single molecule analytical methods capable of visualizing and quantitatively examining the function(s) of the human MMR components on naked DNA and chromatin. These studies will provide a platform for understanding the detailed function(s) of the core MMR components in excision repair, damage signaling, drug resistance and tumorigenesis